We have discovered a frequent mutation in the estrogen receptor ER? in over 30% of hyperplasias of the breast, and strikingly in 50% of invasive breast cancers. This mutation replaces arginine for lysine at residue 303 (K303R), which greatly enhances sensitivity to very low levels of estrogen. Furthermore, the mutant ER? causes the antiestrogen tamoxifen (Tam) to act as an agonist rather than an antagonist, which would lead to Tam resistance in tumors. (Others have found that signaling from overexpressed HER2 causes wild type (WT) ER? to behave the same way.) Furthermore, upon estrogen withdrawal, our mutant-expressing tumors re- gress but then regrow, suggesting that the mutation also confers resistance to therapies such as aromatase inhibitors (AIs). The mutation enhances phosphorylation of ER? at the adjacent S305 site by the intracellular signaling kinases AKT and PKA, and S305 phosphorylation enhances phosphorylation at S118 and may affect the Y537 site phosphorylated by c-Src, since the mutation also enhances ER? cross-talk with c-Src activity. Collectively, our data suggest that the hinge domain where the mutation and the S305 phosphorylation site reside regulates interactions among ER?'s functional domains as well as with coregulators and target promoters. We therefore hypothesize that the mutation adapts ER? for enhanced reception of intra-cellular signal transduction, which alters ER? activity and confers resistance to hormonal therapies. To test this hypothesis and to explore its implications for breast cancer biology and especially for clinical resistance to endocrine therapies, we propose: (1) To determine the interaction of the K303R mutation and S305 phosphorylation of ER? in hormone resistance. We will generate MCF-7 WT and K303R ER? lines with additional ER??mutations to either eliminate or mimic activation of the phosphorylation sites at S305, S118, and Y537. Responses to Tam and estrogen will be determined in vitro and in xenografts, signaling from constitutively activated PKA. For exploring effects on AI therapy, aromatase cDNA will be stably introduced into the various double mutant lines. (2) To determine the gene-selective effects of S305 ER? phosphorylation events. To identify relationships between intracellular kinase signaling, ER? function, and hormone resistance, we will perform expression microarray analyses of our models from Aim 1. Tam agonist activities on selected genes will be confirmed with luciferase reporters, and immunoblot analysis will use phosphorylation-specific ER? antibodies. To discover genes induced by Tam's agonist effects we will employ ChIP-on-chip assays, with the goal of identifying potential targets for intervention in resistance. (3) To determine the impact of the K303R mutant and receptor phosphorylation on clinical hormone resistance. Tumors from women treated with Tam, with long-term clinical follow-up, will be sequenced for the K303R mutation to determine if the mutation affects Tam response, and phosphorylation at S305 will be assessed by IHC. Finally, we will determine if the mutant is associated with the luminal B expression profile subset and its poor clinical outcome. PROJECT NARRATIVE We discovered the K303R ER? mutation in breast hyperplasias and determined it was present in invasive cancer and associated with biological features of poor clinical outcome. The mutation confers an estrogen hypersensitive, tamoxifen-resistant phenotype to breast cancer cells. We will test whether the mutation is associated with poor response to tamoxifen and the aromatase inhibitor anastrazole.